Methods for introducing nucleic acids into mammalian cells using imidazolium lipids

ABSTRACT

Methods for introducing nucleic acids into mammalian cells are provided which use imidazolium lipids. The imidazolium lipids have the formula:                    
     wherein R 1  and R 2  each independently represent a C 8 -C 24  saturated or unsaturated hydrocarbon chain, uninterrupted or interrupted by from 1 to 3 heteroatom moieties selected from —O—, —S—, —NH— and —NR—; X represents —CH 2 —, —O—, —S—, —NH— or —NR—; wherein R is a lower alkyl group having from 1 to 4 carbon atoms; n is an integer of from 1 to 2; and A −  is an anion.

This application is a divisional of application Ser No. 09/183,634,filed Oct. 30, 1998, now U.S. Pat No. 6,121,457 which claims priorityfrom Ser. No. 60/065,859 filed Nov. 14, 1997, the disclosures of eachbeing incorporated by reference.

FIELD OF THE INVENTION

This invention relates to novel cationic lipids which contain a five- orsix-member nitrogen containing ring and attached fatty acid chains whichare stable to hydrolysis. The lipids are useful in the preparation ofliposomes and other lipid vesicle carriers of nucleic acids and othersubstances, for delivery to cells.

BACKGROUND OF THE INVENTION

The introduction of genetic material into a cell can facilitateexpression of an encoded protein to complement a deficient or defectiveprotein. The use of such technology allows for the treatment of diseaseas well as production of certain proteins in an in vitro application.

One method of introducing nucleic acids into a cell is mechanically,using direct microinjection. However this method is labor-intensive and,therefore, only practical for transfecting small numbers of cells suchas eukaryotic germline cells for the production of transgenic systems.To be effective in treating a disease, a nucleic acid-based therapytypically must enter many cells.

Gene transfer entails distributing nucleic acids to target cells andthen transferring the nucleic acid across a target cell membrane intactand, typically, into the nucleus in a form that can function in atherapeutic manner. In vivo gene transfer is complicated by seruminteractions, immune clearance, toxicity and biodistribution, dependingon the route of adminstration.

The in vivo gene transfer methods under study in the clinic consistalmost entirely of viral vectors. Although viral vectors have theinherent ability to transport nucleic acids across cell membranes andsome can integrate exogenous DNA into the chromosomes, they can carryonly limited amounts of DNA and also pose risks. One such risk involvesthe random integration of viral genetic sequences into patientchromosomes, potentially damaging the genome and possibly inducing amalignant transformation. Another risk is that the viral vector mayrevert to a pathogenic genotype either through mutation or geneticexchange with a wild-type virus.

More recently, cationic lipids have been used to deliver nucleic acidsto cells, allowing uptake and expression of foreign genes both in vivoand in vitro. While the mechanism by which cationic lipid carriers actto mediate transfection is not clearly understood, they are postulatedto act in a number of ways with respect to both cellular uptake andintracellular trafficking. Some of the proposed mechanisms by whichcationic lipids enhance transfection include: (i) compacting the DNA,protecting it from nuclease degradation and enhancing receptor-mediateduptake, (ii) improving association with negatively-charged cellularmembranes by giving the complexes a positive charge, (iii) promotingfusion with endosomal membranes facilitating the release of complexesfrom endosomal compartments, and (iv) enhancing transport from thecytoplasm to the nucleus where DNA may be transcribed. When used for invivo delivery, the role of the cationic lipid carriers is furthercomplicated by the interactions between the lipid-nucleic acid complexesand host factors, e.g., the effects of the lipids on binding of bloodproteins, clearance and/or destabilization of the complexes.

Typically, cationic lipids are mixed with a non-cationic lipid, usuallya neutral lipid, and allowed to form stable liposomes, which liposomesare then mixed with the nucleic acid to be delivered. The liposomes maybe large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) orsmall unilamellar vesicles (SUVs). The liposomes are mixed with nucleicacid in solution, at concentrations and ratios optimized for the targetcells to be transfected, to form cationic lipid-nucleic acidtransfection complexes. Alterations in the lipid formulation allowpreferential delivery of nucleic acids to particular tissues in vivo.PCT patent application numbers WO 96/40962, WO 96/40963. Certainpreformed cationic liposome compositions are available, such asLIPOFECTIN® and LIPOFECTAMINE®. Another method of complex formationinvolves the formation of DNA complexes with mono- or poly-cationiclipids without the presence of a neutral lipid. These complexes areoften not stable in water. Additionally, these complexes are adverselyaffected by serum (see, Behr, Acc. Chem. Res. 26:274-78 (1993)). Anexample of a commercially available poly-cationic lipid is TRANSFECTAM®.

While the use of cationic lipid carriers for transfection is now wellestablished, structure activity relationships are not well understood.It is postulated that different lipid carriers will affect each of thevarious steps in the transfection process (e.g., condensation, uptake,nuclease protection, endosomal release, nuclear trafficking, anddecondensation) with greater or lesser efficiency, thereby making theoverall transfection rate difficult to correlate with lipid structures.Thus, alterations in either the cationic or neutral lipid component donot have easily predictable effects on activity. For the most part,therefore, improvements to known cationic lipid-mediated deliverysystems are dependent on empirical testing. When intended for in vivotransfection, new lipids and lipid formulations should be screened invivo to accurately predict optimal lipids and formulations fortransfection of target cells.

More recently, new cationic lipids have been prepared which exhibitexcellent transfection properties when formulated with nucleic acids.See WO 95/14380, the disclosure of which is incorporated herein byreference. The compositions provided in WO 95/14380 are metabolizable inanimal cells to components that are typically endogenous to the cells.Despite the properties associated with the novel cationic lipids, thereexists a need for cationic lipids which are more hydrolytically stableand which can be formulated into suitable transfection compositions. Thepresent invention provides such cationic lipids, along with methods fortheir preparation and use in lipid-based compositions.

Relevant Literature

Cationic lipid carriers have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., (1987) Proc. Natl. Acad. Sci.(USA), 84:7413-7416); mRNA (Malone et al., (1989) Proc. Natl. Acad. Sci.(USA) 86:6077-6081); and purified transcription factors (Debs et al.,(1990) J. Biol. Chem. 265:10189-10192), in functional form. Literaturedescribing the use of lipids as carriers for DNA include the following:Zhu et al., (1993) Science, 261:209-211; Vigneron et al., (1996) Proc.Natl. Acad. Sci. (USA), 93:9682-9686; Hofland et al., (1996) Proc. Natl.Acad. Sci. (USA), 93:7305-7309; Alton et al., (1993) Nat. Genet.5:135-142; von der Leyen et al., (1995) Proc. Natl. Acad. Sci. (USA),92:1137-1141; See also Stribling et al., (1992) Proc. Natl. Acad. Sci(USA) 89:11277-11281, which reports the use of lipids as carriers foraerosol gene delivery to the lungs of mice. For a review of liposomes ingene therapy, see Lasic and Templeton, (1996) Adv. Drug Deliv. Rev.20:221-266.

The role of helper or neutral lipids in cationic lipid-mediated genedelivery is described in Felgner et al., (1994) J. Biol. Chem. 269(4):2550-2561 (describing improved transfection using DOPE); and Hui et al.,(1996) Biophys. J. 71: 590-599. The effect of cholesterol on liposomesin vivo is described in Semple et al., (1996) Biochem. 35(8): 2521-2525.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds having theformula:

The symbols in this formula have the following meanings:

R¹ and R² each independently represent a C₈-C₂₄ saturated or unsaturatedhydrocarbon, alkyl or acyl, chain, which is optionally interrupted byfrom 1 to 3 heteroatom moieties, such as —O—, —S—, —NH— and —NR⁶—;

R³ and R⁴ each independently represent H or a C₁-C₅ saturated orunsaturated hydrocarbon chain;

X¹ represents —CH₂—, —O—, —S—, —NH— or —NR⁷—;

X² represents —CH₂—, —O—, —S—, —NH— or —NR⁸—;

R⁵ represents H, R⁹ or —(CH₂)_(m)—Y;

Y represents —OH, —NH₂, —NHR¹⁰ or —NR¹¹R¹²;

R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² each independently represent an alkylor acyl group having from 1 to 4 carbon atoms;

m represents an integer from 1 to 5;

n represents 0 or 1;

A⁻ represents an anion, preferably chloride or citrate; and

t is an integer equal to the number of positive charges borne by themolecule.

In compounds according to Formula (I), wherein one or more of R¹, R²,X¹, X² and/or Y comprise an amine group, one or more of the amine groupscan be a quaternary amine group. Quaternization can be effected byprotonation or other mechanisms known in the art.

In another aspect, the present invention provides lipid vesiclecompositions which comprise a compound of the formula provided above.

In yet another aspect, the present invention provides lipid:nucleic acidcomplexes which comprise a nucleic acid, typically as DNA, and acompound of Formula (I), above.

In still another aspect, the present invention provides a method fortransfecting mammalian cells, comprising contacting the cells with acomposition comprising an expression cassette and a compound of Formula(I), (II), or (III).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a synthesis scheme for a compound of Formula (I) inwhich X¹ is —CH₂—, X² is —O— and R⁵ is H.

FIG. 2 provides a synthesis scheme for a compound of Formula (I) inwhich X¹ and X² are —O— and R⁵ is H.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

The abbreviations used herein have the following meanings: DOTIM,1-[2-(9(z))-octadecenoyloxy)ethyl]-2-(8(z)-heptadecenoyl0-3-(2-hydroxyethyl)imidazoliniumchloride; EDTA, ethylenediaminetetraacetic acid; CAT, chloramphenicolaminotransferase; D5W, 5% dextrose in water; HCMV, humancytomegalovirus; CHOL, cholesterol; DLPE,dilauroylphosphatidylethanolamine; IV, intravenous; CBC, complete bloodcount;

Embodiments of the Invention Compounds

As noted in the Summary, the present invention provides novel cationiclipids which are hydrolytically stable. As a result, these lipids areuseful in the preparation of lipid vesicle carriers, or lipid-nucleicacid complexes which are used for transfection of cells.

The cationic lipids of the present invention are represented by Formula(I):

In this formula, R¹ and R² each independently represent a C₈-C₂₄saturated or unsaturated hydrocarbon chain, which is optionallyinterrupted by from 1 to 3 heteroatom moieties, such as —O—, —S—, —NH—and —NR—. In preferred embodiments, R¹ and R² each independentlyrepresent a C₈-C₂₄ saturated or unsaturated hydrocarbon chain. Examplesof suitable saturated hydrocarbon chains include those chains derivedfrom fatty acids or alcohols, for example, lauryl (C12:0), myristyl(C14:0), palmityl (C16:0), stearyl (C18:0), arachidyl (C20:0), andbehenyl (C22:0). Examples of suitable unsaturated hydrocarbon chainsinclude, for example, oleyl (C18:1,cis-9), linoleyl (C18:2,cis-9,12),elaidyl (C18:1,trans-9), linolelaidyl (C18:2,trans-9,12), eicosenyl(C20:1,cis-11), and the like. In each of the above, the configuration isprovided as, for example, (C20:1,cis-11), indicating a twenty carbonchain having a single cis double bond between the eleventh and twelfthcarbon atoms (when counting the carbon atoms in the conventional mannerfor fatty acids). While examples are provided for saturated andunsaturated hydrocarbon chains having an even number of carbon atoms,the invention is not so limited. A variety of methods are available tothe skilled chemist for a one-carbon homologation or degradation toprovide hydrocarbon chains having an odd number of carbon atoms.Additionally, due to the synthetic scheme provided in FIG. 1 anddiscussed below, R² will have an odd number of carbon atoms whensynthesis is carried out using commercially available fatty acids suchas oleic acid, myristic acid, palmitic acid, and the like. Stillfurther, R¹ will typically have an odd number of carbon atoms whensynthesis (according to FIG. 1) is carried out beginning with a fattyalcohol having an even number of carbon atoms (to produce compounds ofFormula (I) in which X is —CH₂—).

In one group of preferred embodiments, R¹ and R² will each independentlybe a C₁₂-C₂₀ saturated hydrocarbon chain. In another group of preferredembodiments R¹ and R² will each independently be a C₁₂-C₂₀ unsaturatedhydrocarbon chain. Particularly preferred embodiments, within the groupof unsaturated hydrocarbon chains, are those which contain a singledouble bond in the cis orientation (e.g., oleyl, elaidyl, and thosehaving one more or one less carbon atoms).

R³ and R⁴ are each independently H or a C₁-C₅ saturated or unsaturatedhydrocarbon chain. Examples of hydrocarbon chains having from 1 to 5carbon atoms include, but are not limited to, methyl, ethyl, propyl,cyclopropyl, isopropyl, n-butyl, i-butyl, t-butyl, pentyl, cyclopentyl,cis-2-pentene, trans-2-pentene and the like.

In some embodiments, various groups, known to those of skill in the art,may be added to the compounds to enhance properties such as watersolubility, targetting of particular cell surfaces, or fusogenicity. Forexample, poly(ethyleneglycol) can be attached to a hydroxyl group at R₅.

In Formula (I) above, X¹ represents a linking group such as —CH₂—, —O—,—S—, —NH— or —NR⁷—. Preferably, X is —CH₂— or —O—, more preferably—CH₂—.

The compounds of the invention comprise both 5- and 6-membered ringheterocycles. Thus, the subscript n in Formula (I) is either 0 or 1,preferably, 0. Additionally, the dashed arc and + sign within thenitrogen heterocycle are meant to indicate the presence of a double bondbetween either of the nitrogen atoms in the ring and the single carbonatom which joins the two nitrogen atoms. As a result, a positive charge(+) is associated with one or the other of the nitrogen atoms, dependingon the position of the double bond (i.e., the positive charge isassociated with the nitrogen atom which forms one end of the doublebond).

X² is a linking group which is either —CH₂—, —O—, —S—, —NH— or —NR⁸—.Preferably, X is —CH₂— or —O—, more preferably —O—.

R⁵ is H, R⁹ or —CH₂)_(m)—Y, preferably H.

Y is —OH, or an amino or alkylamino group such as —NH², —NHR¹⁰,—NR¹¹R¹², preferably —OH.

The alkyl substituents on the amino groups, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ andR¹², each independently represent an alkyl group having from 1 to 4carbon atoms. Examples of hydrocarbon chains having from 1 to 4 carbonatoms include, but are not limited to, methyl, ethyl, propyl,cyclopropyl, isopropyl, n-butyl, i-butyl, t-butyl and cyclobutyl. Thealkyl groups can be either saturated or unsturated. In preferredembodiments, the alkyl groups are methyl or ethyl, more prefereablymethyl. In addition, in some embodiments, these amines can bequaternized for example, by methylation.

The symbol A— represents an anion, preferably a pharmaceuticallyacceptable anion. As used herein, the term “pharmaceutically acceptableanion” refers to anions of organic and inorganic acids which providenon-toxic salts in pharmaceutical preparations. Examples of such anionsinclude, but are not limited to, chloride, bromide, sulfate, phosphate,acetate, benzoate, citrate, glutamate, and lactate. The preparation ofpharmaceutically acceptable salts is described in Berge, et al., JPharm. Sci. 66:1-19 (1977), incorporated herein by reference.Preferably, A— is chloride, bromide or citrate.

In one group of particularly preferred embodiments, R¹ and R² eachindependently represent a C₁₂-C₁₈ unsaturated hydrocarbon chain, X¹ is—O—, X² is —OH or —CH₂)₂—OH and n is 0. In another group of particularlypreferred embodiments, R¹ represents a C₁₈-unsaturated hydrocarbonchain, R² represents a C₁₇ unsaturated hydrocarbon chain, X¹ is —O—, X²is —OH and n is 0.

In another group of particularly preferred embodiments, X¹ is —CH₂—.Within this group of embodiments, R¹ will preferably have an odd numberof carbon atoms and contain one double bond having a cis configuration.Also preferred are those embodiments in which R² is a saturatedhydrocarbon chain having an odd number of carbon atoms, or anunsaturated hydrocarbon chain having an odd number of carbon atoms withone double bond having a cis configuration. Most preferred in this groupof embodiments are the compounds of Formulas (II) and (III).

Preparation of the Imidazolinium Lipids

Preparation of lipids of Formula (I) can be carried out according to theoutline provided in FIG. 1. Briefly, a fatty alcohol is converted to asulfonate ester (an activated leaving group) by treatment with, forexample, methanesulfonyl chloride or p-toluenesulfonyl chloride toprovide a compound a. Reaction of compound a with2-(2-aminoethylamino)ethanol provides compound b. Protection of each ofthe amino groups as their t-butyl carbamate can be accomplished withreagents such as (t-Boc)₂O or BOC-ON (from Aldrich Chemical Co.,Milwaukee, Wis., USA) to provide compound c. Following protection of theamino groups, the hydroxyl group can be acylated with an appropriateacid chloride, typically a fatty acid chloride to provide compound dwhich is then deprotected to provide compound e. Heating compound e in asuitable polar solvent (e.g., ethylene glycol) produces an imidazoliniumring (or the related six-member ring) with concomitant loss of water,and provides target compound f. As can be seen from FIG. 1, thelimitations on R¹ and R² in Formula (I) will depend only on theavailability of fatty alcohols and fatty acids. Accordingly, compoundsof Formula (I) are readily formed in which R² has an odd number ofcarbon atoms, optionally with one or more sites of unsaturation.Similarly, for those compounds of Formula (I) in which X is —CH₂—, R¹will have an odd number of carbon atoms when the starting material isone of the readily available fatty alcohols (e.g., stearyl alcohol andoleyl alcohol). Preparation of compounds in which R¹ or R² have an evennumber of carbon atoms can be accomplished by suitable conversion of thestarting fatty acids or alcohols to their longer or shorter homologs.Additionally, position and geometry (cis or trans) of any double bondspresent can also be altered in the starting materials using knownmethods for isomerization of double bonds as well as positional transferof double bonds.

The salt formed using the procedures in FIG. 1 is a chloride salt.Replacement of the chloride ion with another anion can be accomplishedusing, for example, ion exchange resins which are equilibrated with thedesired anion.

Other compounds of Formula (I) can be prepared by methods outlined inFIG. 2. As outlined in FIG. 2, compounds of Formula (I) in which X is—O— can be prepared from fatty alcohols, fatty acids, andN,N′-di(2-hydroxyethyl)-ethylene diamine. Briefly, the amino groups ofN,N′-di(2-hydroxyethyl)-ethylene diamine are protected as their Boccarbamates to provide compound g, which is then monoalkylated with anactivated fatty alcohol sulfonate ester (compound a) to form compound h.Acylation of the remaining alcohol moiety is accomplished by treatmentof compound h with a fatty acid chloride to provide compound i.Conversion of compound i to target compound k is carried out by firstremoving the Boc protecting groups then heating the resultant compound jin a polar solvent to effect the cyclization to compound k.

Lipid Vesicle Compositions

In another aspect, the present invention provides lipid vesiclecompositions which contain a lipid of Formula (I), above. The lipidvesicle compositions will typically be in the form of liposomes (e.g.,unilammelar vesicles, multilammelar vesicles), or other lipid bilayerforms. The cationic lipids described above can be used alone or combinedwith other lipids in the preparation of compositions useful inintracellular delivery systems (e.g., transfection systems), or otherconventional drug delivery systems.

A variety of other lipids are suitable for use in the presentcompositions. As used herein, the term “lipid” refers to any suitablematerial resulting in a bilayer in aqueous solution such that ahydrophobic portion of the lipid material orients toward the bilayerwhile a hydrophilic portion orients toward the aqueous phase.Hydrophilic characteristics derive from the presence of phosphate,carboxylic, sulfato, amino, sulfhydryl, nitro, and other like groups.Hydrophobicity is conferred by the inclusion of groups that include, butare not limited to, long chain saturated and unsaturated aliphatichydrocarbon groups, with such groups being optionally substituted by oneor more aromatic, cycloaliphatic or the preferred lipids for use inconjunction with the cationic lipids of Formula (I) arephosphoglycerides, sphingolipids and sterols (e.g. cholesterol),representative examples of which include diacyl derivatives ofphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol and phosphatidic acid (e.g., palmitoyloleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine anddilinoleoylphosphatidylcholine) and the related lysophosphatidylcholineand lysophosphatidylethanolamine. Still other lipids are useful, such assphingolipid and glycosphingolipid families. Additionally, the lipidsdescribed above may be mixed with other lipids (co-lipids) includingtriglycerides and sterols (e.g., cholesterol).

When the lipid vesicle compositions are in the form of liposomes, thecompounds of Formula (I) will typically be combined with other lipids,including neutral lipids, zwitterionic lipids, anionic lipids or othercationic lipids known to those of skill in the art. Preferably, thecompositions will comprise other neutral lipids or zwitterionic lipids(e.g., cholesterol, dilaurylphosphatidylethanolamine). Particularlypreferred co-lipids include DOPE, DLPE and cholesterol. For systemicdelivery of nucleic acids, cholesterol is the preferred co-lipid.

In general, less saturated lipids are more easily sized, particularlywhen the liposomes must be sized below about 0.3 microns. In one groupof embodiments, lipids containing saturated fatty acids with carbonchain lengths in the range of C₁₄ to C₂₂ are preferred. In another groupof embodiments, lipids with mono- or di-unsaturated fatty acids withcarbon chain lengths in the range of C₁₄ to C₂₂ are used. Additionally,lipids having mixtures of saturated and unsaturated fatty acid chainscan be used.

A number of methods are available for preparing liposome compositions(see, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467(1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, the textLiposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983,Chapter 1, and Hope, et al., Chem. Phys. Lip. 40:89 (1986), all of whichare incorporated herein by reference). One method produces multilamellarvesicles of heterogeneous sizes. In this method, the vesicle-forminglipids are dissolved in a suitable organic solvent or solvent system anddried under vacuum or an inert gas to form a thin lipid film. Ifdesired, the film may be redissolved in a suitable solvent, such astertiary butanol, and then lyophilized to form a more homogeneous lipidmixture which is in a more easily hydrated powder-like form. This filmis covered with an aqueous buffered solution and allowed to hydrate,typically over a 15-60 minute period with agitation. The sizedistribution of the resulting multilamellar vesicles can be shiftedtoward smaller sizes by hydrating the lipids under more vigorousagitation conditions.

Following liposome preparation, the liposomes may be sized, for example,by extrusion, sonication, filtration or emulsification to achieve adesired size range and a relatively narrow distribution of liposomesizes, which is particularly desirable for in vivo delivery of nucleicacids. A size range of about 0.2-0.4 microns allows the liposomesuspension to be sterilized by filtration through a conventional filter,typically a 0.22 micron filter. The filter sterilization method can becarried out on a high through-put basis if the liposomes have been sizeddown to about 0.2-0.4 microns.

Techniques for sizing liposomes to a desired size, include sonication(see, U.S. Pat. No. 4,737,323, incorporated herein by reference),extrusion through small-pore polycarbonate membranes or an asymmetricceramic membrane, and homogenization. Homogenization relies on shearingenergy to fragment large liposomes into smaller ones. In a typicalhomogenization procedure, multilamellar vesicles are recirculatedthrough a standard emulsion homogenizer until selected liposome sizes,typically between about 0.1 and 0.5 microns, are observed. In any of themethods, the liposome size distribution can be monitored by conventionaldynamic laser light scattering.

The lipid vesicle compositions will, in some embodiments, have anattached targeting moiety. For example, a ligand binding specifically toa receptor on a particular target cell type can be used to targetdelivery of the lipid carrier (with, e.g., the DNA or antibiotic ofinterest) to the desired target cells. Alternatively, the activecompound may be a peptide or other small molecule designed to regulateintracellular trafficking of the delivered substance, e.g., triggeringendosomal release or transport into the nucleus using a nuclearlocalizing sequence.

The active compounds bound to the lipid mixture can vary widely, fromsmall haptens (molecular weights of about 125 to 2000) to antigens(molecular weights ranging from around 6000 to 1 million). Of particularinterest are proteinaceous ligands that bind to and are internalized byspecific complementary binding partners on cell surfaces. Illustrativeactive compounds include cytokines, interferons, hormones,asialeglycoprotein antibodies to cell surface receptors or othermolecules, and fragments of such compounds that retain the ability tobind to the same cell surface binding partners that bind the original(non-fragment) molecules.

The number of active compounds bound to a lipid carrier will vary withthe size of the complex, the size of the compound, the binding affinityof the molecule to the target cell receptor or ligand, and the like.Usually, the bound active molecules will be present in the lipid mixturein from about 0.001 to 10 mole percent, more usually from about 0.01 to5 mole percent based on the percent of bound molecules to the totalnumber of molecules available in the mixture for binding.

Lipid-Nucleic Acid Compositions

In another aspect, the present invention provides lipid-nucleic acidcompositions in which the lipid portion comprises at least one cationiclipid of Formula (I). Preferably, the cationic lipids of Formula (I) arethose in which X is —CH₂—. Other preferred embodiments for the cationiclipid and, optionally, co-lipids are as described above.

The nucleic acids which are useful in the present invention aretypically nucleotide polymers having from 10 to 100,000 nucleotideresidues. Typically, the nucleic acids are to be administered to asubject for the purpose of replacing or enhancing the expression of acellular protein and may also encode an RNA molecule, e.g. antisense RNAor ribozyme, which will inhibit an undesired cellular activity, e.g. ina virus-infected cell or tumor cell. Additionally, the nucleic acid cancarry a label (e.g., radioactive label, fluorescent label orcalorimetric label) for the purpose of providing clinical diagnosisrelating to the presence or absence of complementary nucleic acids.Accordingly, the nucleic acids, or nucleotide polymers, can be polymersof nucleic acids including genomic DNA, cDNA, mRNA or oligonucleotidescontaining nucleic acid analogs, for example, the antisense derivativesdescribed in a review by Stein, et al., Science 261:1004-1011 (1993) andin U.S. Pat. Nos. 5,264,423 and 5,276,019, the disclosures of which areincorporated herein by reference. Still further, the nucleic acids mayencode transcriptional and translational regulatory sequences includingpromoter sequences and enhancer sequences.

The nucleotide polymers can be single-stranded DNA or RNA, ordouble-stranded DNA or DNA-RNA hybrids. Examples of double-stranded DNAinclude structural genes including control and termination regions, andself-replicating systems such as plasmid DNA.

Single-stranded nucleic acids include antisense oligonucleotides(complementary to DNA or RNA), ribozymes and triplex-formingoligonucleotides. In order to increase stability, some single-strandednucleic acids will preferably have some or all of the nucleotidelinkages substituted with stable, non-phosphodiester linkages,including, for example, phosphorothioate, phosphorodithioate,phosphoroselenate, or O-alkyl phosphotriester linkages.

The nucleic acids used in the present invention will also include thosenucleic acids in which modifications have been made in one or more sugarmoieties and/or in one or more of the pyrimidine or purine bases.Examples of sugar modifications include replacement of one or morehydroxyl groups with halogens, alkyl groups, amines, azido groups orfunctionalized as ethers or esters. Additionally, the entire sugar maybe replaced with sterically and electronically similar structures,including aza-sugars and carbocyclic sugar analogs. Modifications in thepurine or pyrimidine base moiety include, for example, alkylated purinesand pyrimidines, acylated purines or pyrimidines, or other heterocyclicsubstitutes known to those of skill in the art.

Multiple genetic sequences can also be used in the present methods.Thus, the sequences for different proteins may be located on the same orseparate plasmids. The gene of interest will be linked to appropriateregulatory elements to provide constitutive or inducible expressionand/or tissue specific expression. Additional elements such asantibiotic-sensitive or nutrient-sensitive regions, may be included asrequired. Non-coding sequences may also be present for various purposesincluding, for example, regulatory elements to achieve appropriateexpression or replication in host cells, or to provide convenientcloning sites.

The nucleic acids used in the present invention can be isolated fromnatural sources, obtained from such sources as ATCC or GenBank librariesor prepared by synthetic methods. Typically, the nucleic acids will beplasmid DNA, which can be grown and purified in large quantities frombacterial cells.

Synthetic nucleic acids can be prepared by a variety of solution orsolid phase methods. Generally, solid phase synthesis is preferred.Detailed descriptions of the procedures for solid phase synthesis ofnucleic acids by phosphite-triester, phosphotriester, and H-phosphonatechemistries are widely available. See, for example, Itakura, U.S. Pat.No. 4,401,796; Caruthers, et al., U.S. Pat. Nos. 4,458,066 and4,500,707; Beaucage, et al., Tetrahedron Lett., 22:1859-1862 (1981);Matteucci, et al., J. Am. Chem. Soc., 103:3185-3191 (1981); Caruthers,et al., Genetic Engineering, 4:1-17 (1982); Jones, chapter 2, Atkinson,et al., chapter 3, and Sproat, et al, chapter 4, in OligonucleotideSynthesis: A Practical Approach, Gait (ed.), IRL Press, Washington D.C.(1984); Froehler, et al., Tetrahedron Lett., 27:469-472 (1986);Froehler, et al., Nucleic Acids Res., 14:5399-5407 (1986); Sinha, et al.Tetrahedron Lett., 24:5843-5846 (1983); and Sinha, et al., Nucl. AcidsRes., 12:4539-4557 (1984) which are incorporated herein by reference.

The compositions in this aspect of the invention can be in the form ofliposome-encapsulated nucleic acids, lipid-bilayer coated nucleic acids,or complexes formed between lipids and nucleic acids.

Preparation of Lipid-Nucleic Acid Compositions

Various lipid-nucleic acid compositions, wherein the lipid portion istypically in the form of liposomes, can be prepared using methods wellknown in the art. Still other compositions are contemplated by thepresent invention such as those which are described as lipid particlesin WO 96/40964, the disclosure of which is incorporated herein byreference.

For those compositions in which the lipid portion is in the form ofliposomes, the lipid vesicle can be prepared by standard methods such asthose described above. The resulting liposomes are mixed with a nucleicacid solution with constant agitation to form the cationic lipid-nucleicacid transfection complexes. The preferred size will vary depending onuse. For example, smaller transfection complexes are preferred foraerosol administration, thereby reducing shear caused by theaerosolization process. Preferred transfection complex size for aerosoladministration is less than 5000 nm, most preferably from 50 to 300 nm.Preferred transfection complex size for intravenous administration isfrom 50 to 5000 nm, most preferably from 100 to 700 nm. Cationiclipid-nucleic acid transfection complexes can be prepared in variousformulations depending on the target cells to be transfected. See, e.g.,WO 96/40962 and WO 96/40963. Helper lipids used in these compositionscan be substituted and evaluated for concentration, DNA-lipid ratio,etc. For example, if DLPE is substituted for cholesterol, resulting inchanges in the physical characteristics of the lipid carrier, it ispreferred that additional formulations be tested empirically to obtainoptimal results. While a range of lipid-nucleic acid complexformulations will be effective in cell transfection, optimum conditionsare determined empirically in the desired experimental system. Lipidcarrier compositions may be evaluated by their ability to deliver areporter gene (e.g. CAT which encodes chloramphenicol acetyltransferase,luciferase, or β-galactosidase) in vitro, or in vivo to a given tissuein an animal, such as a mouse.

For in vitro transfections, the various combinations are tested fortheir ability to transfect target cells using standard molecular biologytechniques to determine DNA uptake, RNA and/or protein production.Typically, in vitro cell transfection involves mixing nucleic acid andlipid, in cell culture media, and allowing the lipid-nucleic acidtransfection complexes to form for about 10 to 15 minutes at roomtemperature. The transfection complexes are added to the cells andincubated at 37° C. for about four hours. The complex-containing mediais removed and replaced with fresh media, and the cells incubated for anadditional 24 to 48 hours.

In vivo, particular cells can be preferentially transfected, dependingon the route of administration, by the use of particular cationic lipidsfor preparation of the lipid carriers, or by altering the cationiclipid-nucleic acid formulation to preferentially transfect the desiredcell types (WO 96/40962). Thus, for example, in circumstances where anegatively charged complex is desired, relatively less cationic lipidwill be complexed to the nucleic acid resulting in a higher nucleicacid: cationic lipid ratio. Conversely, in circumstances where apositively charged complex is desired, relatively more cationic lipidwill be complexed with the nucleic acid, resulting in a lower nucleicacid: cationic lipid ratio. To avoid precipitation, which generallyoccurs around charge neutrality, net positively charged complexes aregenerally prepared by adding nucleic acid to the liposomes, and netnegatively charged complexes are prepared by adding liposomes to thenucleic acid, in either case with constant agitation.

The lipid mixtures are complexed with DNA in different ratios dependingon the target cell type, generally ranging from about 6:1 to 1:20 μgDNA:nmole cationic lipid. For transfection of airway epithelial cells,e.g., via aerosol, intratracheal or intranasal administration, netnegatively charged complexes are preferred. Thus, preferred DNA:cationiclipid ratios are from about 10:1 to about 1:20, preferably about 3:1.For intravenous administration, preferred DNA:cationic lipid ratiosrange from about 1:3.5 to about 1:20 μg DNA: nmole cationic lipid, mostpreferably, about 1:6 to about 1:15 μg DNA: nmole cationic lipid.Additional parameters such as nucleic acid concentration, buffer typeand concentration, etc., will have an effect on transfection efficiency,and can be optimized by routine experimentation by a person of ordinaryskill in the art. Preferred conditions are described in the Examplesthat follow.

Non-lipid material, (such as biological molecules being delivered to ananimal or plant cell or target-specific moieties) can be conjugated tothe lipid carriers through a linking group to one or more hydrophobicgroups, e.g., using alkyl chains containing from about 12 to 20 carbonatoms, either prior or subsequent to vesicle formation. Various linkinggroups can be used for joining the lipid chains to the compound.Functionalities of particular interest include thioethers, disulfides,carboxamides, alkylamines, ethers, and the like, used individually or incombination. The particular manner of linking the compound to a lipidgroup is not a critical part of this invention, as the literatureprovides a great variety of such methods. Alternatively, some compoundswill have hydrophobic regions or domains which will allow theirassociation with the lipid mixture without covalent linking to one ormore lipid groups.

Administration of Lipid-Nucleic Acid Compositions to a Host

Following formation of the lipid-nucleic acid compositions, thecompositions (or complexes) can be contacted with the cells to betransfected. Contact between the cells and the lipid-nucleic acidcomplexes, when carried out in vitro, will take place in a biologicallycompatible medium. The concentration of complexes can vary widelydepending on the particular application, but is generally between about1 μmol and about 10 mmol. Treatment of the cells with the lipid-nucleicacid complexes will generally be carried out at physiologicaltemperatures (about 37° C.) for periods of time of from about 1 to 48hours, preferably of from about 2 to 4 hours. For in vitro applications,the delivery of nucleic acids can be to any cell grown in culture,including primary cell and immortalized cell lines, whether of plant oranimal origin, vertebrate or invertebrate, and of any tissue or type. Inpreferred embodiments, the cells will be animal cells, more preferablymammalian cells, and most preferably human cells.

In one group of preferred in vitro embodiments, a lipid-nucleic acidcomposition suspension is added to 60-80% confluent plated cells. Theconcentration of the suspension added to the cells is preferably of fromabout 0.01 to 0.2 μg/mL, more preferably about 0.1 μg/mL.

Typical applications include using well known transfection procedures toprovide intracellular delivery of DNA or mRNA sequences which code fortherapeutically useful polypeptides. However, the compositions can alsobe used for the delivery of the expressed gene product or proteinitself. In this manner, therapy is provided for genetic diseases bysupplying deficient or absent gene products (i.e., for Duchenne'sdystrophy, see Kunkel, et al., Brit. Med. Bull. 45(3)₂630-643 (1989),and for cystic fibrosis, see Goodfellow, Nature 341:102-103 (1989)).Other uses for the compositions of the present invention includeintroduction of antisense oligonucleotides in cells (see, Bennett, etal., Mol. Pharm. 41:1023-1033 (1992)).

The compositions of the present invention can also be used for thetransfection of cells in vivo, using methods which are known to those ofskill in the art. In particular, Zhu, et al., Science 261:209-211(1993), incorporated herein by reference, describes the intravenousdelivery of cytomegalovirus (CMV)-chloramphenicol acetyltransferase(CAT) expression plasmid using DOTMA-DOPE complexes. Hyde, et al.,Nature 362:250-256 (1993), incorporated herein by reference, describesthe delivery of the cystic fibrosis transmembrane conductance regulator(CFTR) gene to epithelia of the airway and to alveoli in the lung ofmice, using liposomes.

In one group of embodiments, the in vivo administration of thepharmaceutical compositions is carried out parenterally, e.g.,intraarticularly, intravenously, intraperitoneally, subcutaneously, orintramuscularly. More preferably, the pharmaceutical compositions areadministered intravenously by a bolus injection for delivery to vascularendothelial cells, by intraperitomal injection for delivery toperitoneal macrophages or cells lining the peritoneal cavity, or byintramuscular or subcutaneous injection for delivery toantigen-presenting cells for purposes of eliciting an immune response.For example, see Stadler, et al., U.S. Pat. No. 5,286,634, which isincorporated herein by reference. Intracellular nucleic acid deliveryhas also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY,Academic Press, New York. 101:512-527 (1983); Mannino, et al.,Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. DrugCarrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278(1993).

In other embodiments, the pharmaceutical preparations described hereinmay be contacted with the target tissue by direct application of thepreparation to the tissue. The application may be made by topical,“open” or “closed” procedures. By “topical”, it is meant the directapplication of the pharmaceutical preparation to a tissue exposed to theenvironment, such as the skin, oropharynx, external auditory canal, andthe like. “Open” procedures are those procedures that include incisingthe skin of a patient and directly visualizing the underlying tissue towhich the pharmaceutical preparations are applied. This is generallyaccomplished by a surgical procedure, such as a thoracotomy to accessthe lungs, abdominal laparotomy to access abdominal viscera, or otherdirect surgical approach to the target tissue, for example, heart tissueand/or associated vasculature may be transfected with a gene of interestin conjunction with heart surgery, as an adjunct therapy. “Closed”procedures are invasive procedures in which the internal target tissuesare not directly visualized, but accessed via inserting instrumentsthrough small wounds in the skin. For example, the preparations may beadministered to the peritoneum by needle lavage or to the vasculatureusing a balloon catheter. Likewise, the pharmaceutical preparations maybe administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrazamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

In yet other embodiments, the lipid-nucleic acid complexes can beadministered in an aerosol inhaled into the lungs (see, U.S. Pat. No.5,641,662). For a general review of applicable techniques, see Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York.pp.70-71 (1994).

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as humans,non-human primates, dogs, cats, cattle, horses, sheep, and the like.

The invention will be better understood in light of the followingspecific examples, which are merely illustrative and should not beconstrued as limiting the invention in any respect, as will be evidentto those skilled in the art.

EXAMPLES

All of the reagents were purchased from Aldrich Chemical Company(Milwaukee, Wis., USA), except oleyl alcohol and oleoyl chloride, whichwere obtained from Nu-Chek-Prep, Inc. (Elysian, Minn., USA). Allsolvents were purchased from Fisher Scientific.

Example 1

This example illustrates the preparation of Compound 1 according to thegeneral scheme outlined in FIG. 1.

1.1 Oleyl methanesulfoante (compound 1a)

To a mixture of oleyl alcohol (10 g, 37.2 mmol) and pyridine (13.4 mL)in CH₂Cl₂ (150 mL), MeSO₂Cl (4.35 ml, 56.2 mmol) was added in 40 min.The mixture was stirred at 0° C. for 40 minutes and then roomtemperature for 4 h. Ice-water (60 mL) and Et₂O (100 mL) were added andshaken. The organic layer was washed with 0.5 N HCl (2×100 mL), H₂O (100mL), 0.5 N NaHCO₃ (2×50 mL), H₂O (50 mL) and NaCl (50 mL), and driedover MgSO₄. Evaporation under reduced pressure gave 12 g (93.0%) ofoleyl methanesulfonate 1a as a liquid. ¹H NMR (CDCl₃): δ0.81 (t, J=7,3H), 1.22 (m, 22H), 1.67 (m, 2H), 1.93 (m, 4H), (s, 3H), 4.16 (t, J=7,2H), 5.28 (m, 2H).

1.2 N-(2-Hydroxyethyl)-N′-oleyl-ethylenediamine (compound 1b)

A mixture of oleyl methanesulfonate (7.01 g, 20.3 mmol) and2-(2-aminoethylamino) ethanol (21.81 g, 209.7 mmol) was heated at 50° C.for 1.5 h. After cooling to room temperature, 30 mL of NaOH solution(10%) was added, stirred for 30 minutes, and extracted with Et₂O (100ml). The Et₂O solution was washed with H₂O (3×50 mL) and NaCl solution,dried over MgSO₄ and evaporated under reduced pressure to give 8.41 g ofthe crude product 1b which was used for the next step reaction withoutfurther purification.

1.3 N,N′-Di-t-Boc-N-(2-Hydroxyethyl)-N′-oleyl-ethylenediamine (compound1c)

To a solution of crude N-(2-hydroxyethyl)-N′-oleyl-ethylenediamine (1b,8.41 g) in CH₂Cl₂ (80 mL), (t-Boc)₂O (12.5 g, 57.3 mmol) was addeddropwise. The reaction mixture was stirred at room temperatureovernight, and then washed with H₂O, NaCl solution and dried over MgSO₄.The solvent was evaporated under reduced pressure and the residue waspurified by column chromatography (SiO₂, EtOAc/Hexanes, 0˜15%) toproduce 5.63 g (50.0%) of productN,N′-di-t-Boc-N-(2-hydroxyethyl)-N′-oleyl-ethylenediamine, 1c. ¹H NMR(CDCl₃): δ0.88 (t, J=7, 3H), 1.28 (m, 22H), 1.45 (s, 9H), 1.48 (s, 9H),1.50 (m, 2H), 2.00 (m 4H), 3.19 (m, 2H), 3.36 (m, 6H), 3.75 (m, 2H),5.35 (m, 2H).

1.4 compound 1d

At 0° C., oleyl chloride (4.5 g, 85%, 16.63 mmol) was added dropwise toa mixture of 1c above (6.36 g, 11.48 mmol) and Et₃N (2.4 mL, 17 mmol) inCH₂Cl₂ (50 mL). The reaction mixture was stirred at 0° C. for 3 h androom temperature overnight, and then diluted with Et₂O (100 mL), washedwith H₂O (30 mL), 5% citric acid (30 mL), H₂O (2×30 mL), NaHCO₃ (30 mL),NaCl solution (30 mL) and dried over MgSO₄. The solvent was evaporatedunder reduced pressure and the residue was purified on a silica gelcolumn (EtOAc/hexanes, 0˜5%) to give 9.03 g (96.2%) of product 1d. ¹HNMR (CDCl₃): δ0.89 (m, 6H), 1.28 (m, 42H), 1.47 (s, 18H), 1.50 (m, 2H),1.60 (m, 2H), 200 (m, 8H), 2.30 (t, J=7, 2H), 3.16 (m, 2H), 3.32 (m,4H), 3.46 (m, 2H), 4.16 (M, 2H), 5.35 (m, 4H).

1.5 compound 1e

A mixture of compound 1d above (8 g, 9.78 mmol) and 4N HCl/dioxane (32mL) was stirred at room temperature for 6 h and then filtered. The whitesolid was washed with hexanes (3×10 mL) and dried under vacuum to give6.63 g (98.1%) of product 1e. mp 180˜188° C. ¹H NMR (CDCl₃): δ0.89. (m,6H), 1.28 (m, 42H), 1.62 (m, 2H), 1.86 (m, 2H), 2.00 (m, 8H), 2.46 (t,J=7, 2H), 3.03 (m, 2H), 3.42 (m, 2H), 3.59 (m, 2H), 3.67 (m 2H), 4.47(m, 2H), 5.35 (m, 4H).

1.6 Compound 1

A suspension of 1e above (2.0 g, 2.89 mmol) in ethylene glycol (10 mL)was stirred at 150° C. for 1.5 h. After cooling to room temperature, themixture was diluted with CH₂Cl₂ (100 mL), washed with MeOH/saturatedNaCl (1:1, 3×30 mL), NaCl solution, and dried over MgSO₄. Evaporationand column chromatography (SiO₂, EtOAc/Hexanes 0˜40%, MeOH/EtOAc 0˜5%,MeOH/CH₂Cl₂ 0˜5%) gave Compound 1 (0.88 g, 47.8%) as white wax. ¹H NMR(CDCl₃): δ0.89 (m, 6H), 1.28 (m, 42H), 1.52 (m, 2H), 1.57 (m, 2H), 2.02(m, 8H), 2.75 (m, 2H), 3.44 (m, 2H), 3.49 (m, 2H), 3.87 (m, 2H), 4.06(m, 4H), 5.35 (m, 4H). Anal. Calcd for C₄₀H₇₇N₂OCl. 13/4 H₂O: C, 71.86;H, 12.05; N, 4.19; Cl, 5.31. Found: C, 71.77; H, 11.97; N, 4.14; Cl,5.83.

Example 2

This example illustrates the generality of the synthetic method of setforth in FIG. 1. Using this synthetic route, a wide range of compoundsof the invention having diverse hydrocarbon substituents attached to the5-membered heterocyclic ring can be synthesized. The fatty acid chainsutilized in this example are displayed below.

2.1 Synthesis of Alkyl Methansulfonate

To a mixture of alkyl alcohol (37.2 mmol) and pyridine (13.4 mL) inCH₂Cl₂ (150 mL) was added MeSo₂Cl (4.35 mL, 56.2 mmol) over 40 min. Themixture was stirred at 0° C. for 40 minutes and then at room temperaturefor 4 h. Ice/H₂O (60 mL) and Et₂O (100 mL) were added. The organic layerwas removed, washed with 0.5 N HCl (2×100 mL), H₂O (100 mL), 0.5 NNaHCO₃ (2×50 mL), H₂O (50 mL) and NaCl (50 mL). The organic layer wasthen dried over MgSO₄. The solvent was removed by evaporation affordingthe pure methanesulfonate. Yields and analytical data for the compoundssynthesized are provided below.

2.1a. Dodecanyl methansulfonate: Yield, 94%. ¹H NMR (CDCl₃): 0.88 (t,j=7, 3H), 1.26 (m, 18H), 1.75 (m, 2H), 3.01 (s, 3H), 4.26 (t, j=7, 2H).

2.1b. Tetradecanyl methanesulfonate: Yield 95%. ¹H NMR (CDCl₃): 0.88 (t,j=7, 3H), 1.26 (m, 22H), 1.75 (m, 2H), 3.00 (s, 3H), 4.22 (t, j=7, 2H).

2.1c. Hexadecanyl methansulfonate: Yield 93%. ¹H NMR (CDCl₃): 0.88 (t,j=7, 3H), 1.26 (m, 26H), 1.75 (m, 2H), 3.00 (s, 3H), 4.22 (t, j=7, 2H).

2.1d Octadecanyl methansulfonate: Yield 91%. ¹H NMR (CDCl₃): 0.89 (t,j=7, 3H), 1.27 (m, 30H), 1.75 (m, 2H), 3.01 (s, 3H), 4.23 (t, j=7, 2H).

2.2 Synthesis of N-(2-hydroxyethyl)-N′-alkylethylenediamine

A stirred mixture of alkyl methanesulfonate (20.3 mmol) and2-(2-aminoethylamino)ethanol (21.81 g, 209.7 mmol) was heated at 50° C.for 1.5 h. After cooling to room temperature, 10% NaOH, (30 mL) wasadded. The mixture was stirred for 30 min. then extracted with Et2O (100mL). The Et₂O solution was washed with H₂O (3×50 mL) and NaCl. Theorganic layer was dried (MgSO₄) and the solvent was removed byevaporation. The crude product was used without any furtherpurification.

2.3 Synthesis ofN,N′-di-t-Boc-N-(2-hydroxyethyl)-N′-alkylethylenediamine

To a solution of N-(2-hydroxyethyl)-N′-alkylethylenediamine in CH2Cl2(80 mL) was added (t-Boc)2O (12.5 g, 57.3 mmol) dropwise. After stirringat room temperature overnight, the mixture was washed with H₂O and NaCl.The organic layer was dried over MgSO₄. The solvent was removed byevaporation and the residue was purified by column chromatography (SiO₂,EtOAc/hexanes, 0-15%) giving the N-t-Boc products.

2.3a. Dodecanyl derivative: Yield 51% (2 steps). ¹H NMR (CDCl₃): 0.86(t, j=7, 3H), 1.25 (m, 22H), 1.44 (s, 9H), 1.47 (s, 9H), 1.51 (m, 2H),3.17 (m, 2H), 3.37 (m, 6H), 3.73 (m, 2H).

2.3b. Tetradecanyl derivative: Yield 46% (2 steps). ¹H NMR (CDCl₃): 0.88(t, j=7, 3H), 1.25 (m, 22H), 1.44 (s, 9H), 1.47 (s, 9H), 1.51 (m, 2H),3.17 (m, 2H), 3.37 (m, 6H), 3.73 (m, 2H).

2.3c. Hexadecanyl derivative: Yield 45% (2 steps). ¹H NMR (CDCl₃): 0.87(t, j=7, 3H), 1.25 (m, 26H), 1.44 (s, 9H), 1.47 (s, 9H), 1.51 (m, 2H),3.16 (m, 2H), 3.36 (m, 6H), 3.72 (m, 2H).

2.3 d. Octadecanyl derivative: Yield 51% (2 steps). ¹H NMR (CDCl₃): 0.87(t, j=7, 3H), 1.25 (m, 30H), 1.44 (s, 9H), 1.46 (s, 9H), 1.51 (m, 2H),3.17 (m, 2H), 3.37 (m, 6H), 3.73 (m, 2H).

2.4 Acylation of derivatives from 2.3

At 0° C., to a mixture of the t-Boc protected diamines (11.48 mmol) andEt₃N (2.4 mL) in CH₂Cl₂ (50 mL), the desired acid chloride (12.63 mmol,1.1 eq.) was added dropwise. After stirring at 0° C. for 3 h and then atroom temperature overnight, the mixture was diluted with Et2O (100 mL),washed with H₂O (30 mL), 5% citric acid (30 mL), H₂O (2×30 mL), NaHCO3(30 mL) and NaCl (30 ml). The organic layer was dried (MgSO₄) and thesolvent was removed by evaporation. The compounds were purified bycolumn chromatography (SiO₂, EtOAc/hexanes, 0-5%).

2.4a. Dodecanyl derivative: Yield 76%. ¹H NMR (CDCl₃): 0.88 (m, 6H),1.26 (m, 34H), 1.46 (s, 18H), 1.51 (m, 2H), 1.61 (m, 2H), 2.30 (t, j=7,2H) 3.16 (m,2H), 3.31 (m, 4H), 3.46 (m, 2H), 4.16 (m, 2H).

2.4b. Tetradecanyl derivative: Yield 77%. ¹H NM (CDCl₃): 0.88 (m, 6H),1.26 (m, 42H), 1.46 (s, 18H), 1.51 (m, 2H), 1.61 (m, 2H), 2.30 (t, j=7,2H), 3.16 (m, 2H), 3.31 (m, 4H), 3.46 (m, 2H), 4.16 (m, 2H)

2.4c. Hexadecanyl derivative: Yield 54%. ¹H NMR (CDCl₃): 0.88 (m, 6H),1.26 (m, 50H), 1.46 (s, 18H), 1.51 (m, 2H), 1.61 (m, 2H0), 2.30 (t, j=7,2H), 3.16 (m, 2H), 3.46 (m, 2H), 4.16 (m, 2H).

2.4d. Octadecanyl derivative: Yield 57%. ¹H NMR (CDCl₃): 0.88 (m, 6H),1.26 (m, 58H), 1.46 (s, 18H), 1.46 (s, 18H), 1.51 (m, 2H), 1.61 (m, 2H),2.30 (t, j=7, 2H), 3.16 (m, 2H), 3.31 (m, 4H), 3.46 (m, 2H), 4.16 (m,2H).

2.5 Removal of t-Boc protecting groups

A mixture of a t-Boc protected acylated diamines and 4 N HCl in dioxane(32 mL) was stirred at room temperature for 6 h then filtered. The whitesolid was washed with hexanes (3×10 mL) and dried under vacuum toproduce the diamine dihydrochloride.

2.5a. Dodecanyl derivative: Yield 93%, MP 188-193° C. ¹H NMR(CDCl₃+MeOD): 0.89 (m, 6H), 1.26 (m, 34H), 1.61 (m, 2H), 1.71 (m, 2H),2.44 (t, j=7, 2H) 3.03 (m, 2H), 3.44 (m, 6H), 4.39 (m, 2H).

2.5b. Tetradecanyl derivative: Yield 93%, MP 200-204° C. ¹H NMR(CDCl₃+D₂O): 0.88 (m, 6H), 1.26 (m, 42H), 1.61 (m, 2H), 1.71 (m, 2H),2.44 (t, j=7, 2H), 3.03 (m, 2H), 3.44 (m, 6H), 4.25 (m, 2H).

2.5c. Hexadecanyl derivative: Yield 92%, MP 208-211° C.: Insoluble in ¹HNMR solvents.

2.5d. Octadecanyl derivative: Yield 82%, MP 211-216° C.: Insoluble in ¹HNMR solvents.

2.6 Cyclization

A suspension of the diamine dihydrochloride (2.0 g) in ethylene glycol(10 mL) was heated to 150° C., with stirring for 1.5 h. After cooling toroom temperature, the mixture was diluted with CH₂Cl₂ (100 mL), washedwith MeOH/saturated NaCl (1:1, 3×30 mL), NaCl and the organic layer wasdried (MgSO₄). The solvent was evaporated and the compounds werepurified by column chromatography (SiO₂, EtOAc/hexanes, 0-40%,MeOH/EtOAc, 0-5%, MeOH/CH₂Cl₂, 0-5%).

2.6a. Dodecanyl derivative: Yield 40%, white wax ¹H NMR (CDCl₃): 0.88(m, 6H), 1.26 (m, 34H), 1.53 (m, 2H), 1.61 (m, 2H), 2.75 (m, 2H), 3.43(m, 2H), 3.49 (m, 2H), 3.86 (m, 2H), 4.06 (m, 2H). Anal. C₂₈H₅₇N₂OCl(1.5 H₂O): Theory C(67.27), H(12.01), N(5.60), Cl(7.11); Found C(67.40),H(11.98), N(5.67), Cl(7.37).

2.6b. Tetradecanyl derivative: Yield 35%, white wax. ¹H NMR (CDCl₃):0.88 (m, 6H), 1.26 (m, 42H), 1.56 (m, 2H), 1.66 (m, 2H), 2.74 (m, 2H),3.43 (m, 2H), 3.49 (m, 2H), 3.86 (m, 2H), 4.06 (m, 4H). Anal.C₃₂H₆₅N₂OCl(H₂O): Theory: C(70.26), H(12.26), N(5.12), Cl(6.50); FoundC(70.06), H(12.27), N(5.18), Cl(6.58).

2.6c. Hexadecanyl derivative: Yield 27%, white wax. ¹H NMR (CDCl₃): 0.88(m, 6H), 1.26 (m, 50H), 1.56 (m, 2H), 1.67 (m, 2H), 2.76 (m, 2H), 3.44(m, 2H), 3.50 (m, 2H), 3.87 (m, 2H), 4.06 (m, 4H). Anal.C₃₆H₇₃N₂OCl(3/5H₂O): Theroy: C(72.57), H(12.46), N(4.70), Cl(5.96);Found C(72.57), H(12.59), N(4.67), Cl(6.03).

2.6d. Octadecanyl derivative: Yield 33%, white wax. ¹H NMR (CDCl₃): 0.87(m, 6H), 1.25 (m, 58H), 1.55 (m, 2H), 1.66 (m, 2H), 2.74 (m, 2H), 3.43(m, 2H), 3.49 (m, 2H), 3.86 (m, 2H), 4.06 (m, 4H). Anal.C₄₀H₈₁N₂OCl(H₂O): Theory C(72.89), H(12.60), N(4.25), Cl(5.39); FoundC(72.87), H(12.59), N(4.25), Cl(5.67).

Example 3

This example illustrates the derivatization of the free alcoholichydroxyl group present in the certain of the compounds of the invention.This derivatization proceeds as displayed in Scheme 1.

Briefly, the final cyclized product is reacted with methane sulfonate toafford a reactive intermediate. This intermediate is then reacted withan amine, such as ethanolamine to provide derivatives analogous to thatshown in Scheme 1.

Example 4

This example provides an evaluation of the cationic lipid prepared inExample 1.

Experimental Protocol

Two mice studies have been performed for the evaluation of cationiclipids of the present invention, in comparison with DOTIM (see, Liu, etal., Nature Biotechnology 15:167-173 (1997)). In brief, the lipidcombinations were tested as carriers for gene transfer by intravenousdelivery in ICR female mice (25 g), and expression was determined usingthe plasmid p4119 containing the CAT reporter gene under the control ofthe HCMV promoter. The lipids were dissolved in a mixture of chloroformand methanol (1:1). Lipid films of cationic and neutral lipid (eithercholesterol or DLPE) at a 1:1 molar ratio were formed with a rotaryevaporator. The films were hydrated with 5% dextrose in water (D5W) atroom temperature and extruded through a series of membranes having poresizes of 400 nm, 200 nm, and 50 nm.

DNA-liposome complexes were prepared at a 1:6 DNA:cationic lipid ratio(mg DNA per μmole cationic lipid) by adding the DNA, in a solution at0.625 mg/mL concentration in D5W to the solution of liposomes, in anequal volume, with constant stirring, using a Hamilton Dilutor 540B(Hamilton, Reno, Nev.). DOTIM:cholesterol was used at a 1:6 DNA:cationiclipid ratio. The DNA solution was 0.3125 mg/mL DNA in D5W. The resultingcomplexes were sized using a Submicron Particle Sizer 370 (Nicomp, SantaBarbara, Calif.). Zeta potential was determined by a Zeta Plus, ZetaPotential Analyzer (Brookhaven Instrurtents Corp.). A total of 5 micewere tested per group. A dose of 62.5 μg p4119 plasmid DNA in 200 μL D5Wwas injected by tail vein per mouse. The lungs were harvested after 24hours and assayed for CAT protein by ELISA assay. Each organ washomogenized in 1.0 mL of 5 mM EDTA/0.25M Tris-HCl pH 7.8 containing 5μg/mL Aprotinin (Boehringer Mannheim, Indianapolis, Ind.), 5 μg/mLLeupeptin (Boehringer Mannheim, Indianapolis, Ind.), and 5 mM PMSF(Boehringer Mannheim, Indianapolis, Ind.). The resulting extracts werecentrifuged and aliquots of the supernatant were removed for proteinanalysis, utilizing a bicinchoninic acid based reagent kit (Pierce,Rockford, Ill.). The remaining supernatant was heat treated at 65° C.for 15 min. The CAT activity assay was performed using 5 μL of heattreated supernatant, 25 μL of 125 μg/mL n-Butyryl CoA (Sigma, St. Louis,Mo.), 50 μL of 5 μCi/mL ¹⁴C-chloramphenicol (DuPont NEN, Boston, Mass. )and 50 μL of 0.25 M Tris-HCl/5 mM EDTA. Samples were incubated at 37° C.for 2 h. An addition of 300 μL of mixed xylenes (Aldrich, Milwaukee,Wis.) was made followed by vortexing and centrifugation at 14K rpm for 5min. The xylene layer was then transferred into 750 μL of 0.25 MTris-HCl/5 mM EDTA, vortexed, and centrifuged at 14K rpm for 5 min. Theupper organic phase was then transferred into scintillation vialscontaining 5 mL of Ready Safe Liquid Scintillation Cocktail (Beckman,Fullerton, Calif.). Samples were counted for 1 min each.

Results

Results for evaluation of Compound 1, Compound 2, and DOTIM versuscontrol (D5W) are provided in Table 1.

TABLE 1 Composition DNA/Lipid Ratio Number Expressed D5W (Control) 1:60/2 DOTIM/CHOL 1:6 5/5 Compound 1/CHOL 1:6 5/5 Compound 1/DLPE 1:6 3/5Compound 2/CHOL 1:6 0/5

Compound 1/Chol showed similar lung CAT expression (2693.94 pg CAT/mgsoluble protein) compared to that of DOTIM/Chol (2072.15 pg CAT/mgsoluble protein). However, in a repeated protocol, the lung expressionof Compound 1/Chol was 400.22 pg CAT/mg soluble protein. This differencemay be due to the variation among different animal studies. Theseresults indicate that Compound 1 retained the in vivo activity of DOTIM.

Both cholesterol and DLPE were used as additional lipids in the miceprotocols since both have previously been shown to be useful helperlipids for delivery to lung endothelium by IV delivery. Compound 1/Cholcompositions showed better activity than Compound 1/DLPE in bothprotocols. Therefore, for IV administration, cholesterol is thepreferred helper lipid for Compound 1.

Compound 2 is the stearoyl derivative of DOTIM, which contains no doublebond. No significant lung expression was observed for Compound 2/Chol inmice, indicating that the double bonds of oleoyl derivatives canincrease the in vivo activity.

Example 3

This example provides an evaluation of Compound 1 as a suitable lipid inDNA:lipid complexes for transfection in rabbits.

3.1 Protocol

Pilot studies were conducted to assess the suitability of rabbits as adevelopment model for delivery of lipid:DNA complexes. The studyconfirmed that rabbits offer the ability to express transfected genes aswell as display some anticipated toxicity endpoints. Under suitableconditions, the rabbit was found to develop a dose-responsive vascularleak syndrome with the added advantage of expressing the CAT reporterprotein. The lung was found to be the target organ for the complexesfollowing IV delivery.

3.1a. Dosing for evaluation of Compound 1

A dosing scheme for 4 rabbits is provided in Table 2.

TABLE 2 Rabbit Material Dose Route/Delivery 1 Compound 1/CAT 2.0 mg/kgIV/0.5 mL/min 2 Compound 1/CAT 1.5 mg/kg IV/0.5 mL/min 3 Compound 1/CAT0.5 mg/kg IV/0.5 mL/min 4 Compound 1 highest vol. IV/0.5 mL/kg

A 2 mL blood sample is collected immediately prior to dose delivery forserum collection and another 2 mL for a CBC/clinical chemistry screen.Sufficient blood is also collected at necropsy to perform a CBC andclinical chemistry screen and preserve serum (minimum of 8 mL).

Approximately 24 hours after dosing the animals are euthanized using abarbiturate overdose. Following euthanasia, body weights are determinedand an examination is made for the presence of pleural fluid. Any fluidpresent is harvested while avoiding blood contamination. Total proteinand albumin levels are determined on the pleural fluid.

Tissues are collected and preserved as follows: lung tissue (8 vials inliquid nitrogen, 2 lobes infused with formalin, and optionally 2Whirlpaks® in dry ice); liver tissue (multiple sites in formalin);kidney tissue (multiple sites in formalin); spleen tissue (multiplesites in formalin); heart tissue (multiple sites in formalin); andmultiple serum samples (6 mL minimum in dry ice).

3.2 Results

Animals received the doses provided in Table 2 with no indication ofdistress. No lesions were noted in animals receiving just the lipid(Compound 1). The animal receiving just the lipid had diffusely redlungs which failed to collapse and no airway foam or excessive pleuralfluid was noted. Very scant (normal) levels of pleural fluid werepresent in all animals. Small amounts of this fluid were collected fromrabbits #1 and #3 for routine protein determinations.

Example 4

The compound of Formula III (Compound 2) was compared to Compound 1 forthe ability to transfect lung cells according to the methods describedin Example 2. Both compounds were formulated with cholesterol as theneutral lipid and plasmid p4119, and complexes injected IV into fivemice each as described in Example 2. The CAT ELISA showed a mean CATexpression level of 853.20 (pg/mg) (+/−571.52) for Compound 2, comparedto a mean of 306.09 (pg/mg) (+/−252.610 for Compound 1.

CAT ELISA (pg/mg or mL) for each of the rabbits was as follows: Rabbit#1, 10244.7 lung and 6665.9 serum; Rabbit #2, 963.8 lung and 1637.1serum; Rabbit #3, 88.5 lung and 0 serum; Rabbit #4, 0 lung and 0 serum.

While the invention has been described in the foregoing description, thesame is to be considered as illustrative and not restrictive incharacter. It is to be understood that only the preferred embodimentshave been described and that all changes and modifications that comewithin the spirit of the invention are desired to be covered.

What is claimed is:
 1. A method for introducing a nucleic acid into amammalian cell, said method comprising contacting said cell with acomposition comprising a nucleic acid and a compound having the formula:

wherein R¹ and R² each independently represent a C₈-C₂₄ saturated orunsaturated hydrocarbon chain, uninterrupted or interrupted by from 1 to3 heteroatom moieties selected from the group consisting of —O—, —S—,—NH— and —NR—; X represents a member selected from the group consistingof —CH₂—, —O—, —S—, —NH— and —NR—; wherein R is a lower alkyl grouphaving from 1 to 4 carbon atoms; n is an integer of from 1 to 2; and A⁻is an anion.
 2. A method in accordance with claim 1, wherein R¹ and R²each independently represent a C₈-C₂₄ saturated or unsaturatedhydrocarbon chain.
 3. A method in accordance with claim 1, wherein R¹and R² each independently represent a C₁₂-C₂₀ saturated hydrocarbonchain.
 4. A method in accordance with claim 1, wherein R¹ and R² eachindependently represent a C₁₂-C₂₀ unsaturated hydrocarbon chain.
 5. Amethod in accordance with claim 1, wherein n is
 1. 6. A method inaccordance with claim 1, wherein R¹ and R² each independently representa C₁₂-C₁₆ unsaturated hydrocarbon chain, X is —O—, and n is
 1. 7. Amethod in accordance with claim 1, wherein R¹ represents aC₁₈-unsaturated hydrocarbon chain, R² represents a C₁₇ unsaturatedhydrocarbon chain, X is —O—, and n is
 1. 8. A method in accordance withclaim 1, wherein R¹ and R² each contain a single double bond in the cisorientation.
 9. A method in accordance with claim 1, wherein saidcompound has the formula:


10. A method in accordance with claim 1, wherein said compound has theformula:


11. A method in accordance with claim 1, wherein said compositionfurther comprises cholesterol.
 12. A method in accordance with claim 1,wherein said composition further comprisesdilauroylphosphatidylethanolamine.
 13. A method in accordance with claim1, wherein said composition is in the form of unilamellar vesicles. 14.A method in accordance with claim 1, wherein said nucleic acid is partof an expression cassette.
 15. A method in accordance with claim 1,wherein said nucleic acid is complexed with liposomes comprising saidcompound.
 16. A method in accordance with claim 1, wherein said nucleicacid encodes a therapeutic protein.
 17. A method in accordance withclaim 1, wherein said nucleic acid encodes a ribozyme.